We identified a novel phospholipid, phosphatidylinositol trisphosphate (PIP3) in activated human neutrophils. It has subsequently been identified in a variety of cell types. The transient and damatic nature of its formation suggests that PIP3 itself and not a metabolite is an intracellular signal. Experiments with inhibitors of an enzyme, PI-3 kinase, which catalyzes the formation of PIP3 suggest that PIP3 plays a role in cell proliferation in dividing cells and oxygen radical formation in neurtophils. Recently, we have found p'p~ is also formed in colonic epithelia stimulated with EGF where its formation has been implicated in the inhibition of chloride secretion. Oxygen radical formation has been identified in a variety of cell types in which its formation is indicatvie of an intracellular signal analagous to nitric oxide. Wherease high levels of oxygen radicals are associated with transcellularly activated inflammatory responses, low levels appear to represent intracellular signals. PIP3 is an intracellular messenger which has been hypothesized to elicit oxygen production in neutrophils. In this study we will test the hypothesis that elevation of PIP3 levels triggers H202 formation in colonic epithelia. PIP3 levels will be elevated by addition of Epidermal Growth factor or addition of exogenous p'p~ to permeabilie cells and hydrogen peroxide will be assessed using the fluorescent indicator, 5-(and 6) caboxxy-2'7'-dichlorodihydrofluorescein diacetate bis ester. Because we predict the increase in fluorescence is likely to be of fairly low intensity and localized due to rapid metabolism of hydrogen peroxide, the sensitivity and resolution of the confocal imageing system will greatly enhance our ability to test this hypothesis. Currently, we are in the process of working out optimal multi-labeling parameters for confocal microscopy.